Olga Greengard

Developmental formation and distribution of arginase in rat tissues

Abstract Quantitative measurement of the arginase in the various rat tissues by a single method shows that it occurs among fetal tissues only in liver and later develops to a high concentration in adult liver and to small but significant concentrations in small intestine, kidney, and pancreas. Its development in the lactating mammary gland to a level comparable to the highest ones in extrahepatic tissues is confirmed. Low but apparently significant levels were found in several transplanted tumors. The developmental formation of arginase in the liver occurred in two distinct steps, prenatally during the late fetal period and postnatally during the third week. Injection of thyroxine (but not of hydrocortisone) enhanced the prenatal accumulation of arginase. Injection of hydrocortisone (but not of thyroxine) to 5–8-day-old rats caused the arginase level to rise in 24 hr to adult levels, normally reached only at 28 days of age. Adrenalectomy at 12 days of age inhibited the accumulation of arginase. The results suggest that thyroid and glucocorticoid hormones successively promote the developmental formation of arginase in the liver during the late fetal and the late suckling periods, respectively.

Aspartate aminotransferase in rat tissues: Changes with growth and hormones

Abstract Levels of the two isozymal forms of aspartate aminotransferase (EC 2.6.1.1) were determined in normal and neoplastic rat tissues. Of the adult tissues with high total aspartate aminostransferase activity heart and muscle contain twice as much soluble than particulate isozyme while in liver and kidney (and tumors whose total activity is low) the particulate isozyme predominates. Mammary gland begins to exhibit aspartate aminotransferase activity after parturition and the soluble isozyme continues to rise throughout lactation. Fetal tissues have generally low aspartate aminotransferase activity; the different rates of its accumulation during gestation and postnatal life determined in liver, kidney and heart. The administration of thyroxine 2 days before birth, or that hydrocortisone 2 days before birth, promotes the accumulation of liver aspartate aminotransferase. Estradiol inhibits the developmental formation of the enzyme in liver and prevents the hydrocortisone induction of soluble aspartate aminostransferase in liver of adult males. The results indicate that the amounts of the two forms of aspartate aminotransferase are subject to different physiological control in different tissues. The response of the liver isozymes to hormones depends on the stage of development and after maturity of the sex of the animal.

Enzymes in intracellular organelles of adult and developing rat brain

Abstract Eighty percent of the hexokinase and about a half of the lactate dehydrogenase, pyruvate kinase, and aldolase activities of adult rat cerebral homogenates is particulate, associated to a large extent, with the sediment (P 2 ) obtained by centrifugation at 17,000 g . Centrifugation of P 2 into sucrose gradients shows that all four enzymes are associated with synaptosomes: their peak concentration coincides with that of glutamate decarboxylase rather than with those of mitochondrial enzymes, glutamate dehydrogenase, and aspartate aminotransferase. After hypoosmotic shock and high-speed centrifugation considerable portions of synaptosomal enzymes are recovered in the supernatant phase; the composition of this fluid, as indicated by the higher specific activity of several enzymes, is different from that of the soluble fraction of whole homogenates. The concentration of the seven enzymes studied is considerably lower in fetal than in adult brain and, in general, a larger fraction of the total is soluble. Preferential accumulation with age in the particulate fraction is especially striking in the case of hexokinase. Between fetal and adult life there are changes in the enzymic composition as well as increases in the amount of the total protein attributable to the synaptosomal fraction. Glutamate decarboxylase and lactate dehydrogenase are the synaptosomal enzymes to rise first (before or at birth), followed by hexokinase and, in the third postnatal week, by aldolase and pyruvate kinase. The upsurge of mitochondrial enzymes (that of glutamate dehydrogenase at term and of aspartate aminotransferase 10 days later) is accompanied by insignificant or small increases in the total protein content of the same fraction. The results indicate that the maturation of subcellular organelles involves a stepwise enrichment with various enzymes; some signs of biochemical differentiation precede and others coincide with the development of cerebral functions known to occur in 2- to 4-wk-old rats.

Distribution of mitochondrial enzymes between the perikaryal and synaptic fractions of immature and adult rat brain

The subcellular distribution of mitochondrial enzymes was studied in cerebral hemispheres of 15-day-old and adult rats. At both ages the synaptosomal fraction contained very little glutamate dehydrogenase (EC 1.4.1.2) but significant amounts of succinate dehydrogenase (EC 1.3.99.1), glutaminase (EC 3.5.1.2), hexokinase (EC 2.7.1.1), malate NADP dehydrogenase (EC 1.1.1.40) and beta-hydroxybutyrate dehydrogenase (EC 1.1.1.30). In immature brain, in the fraction enriched with free (perikaryal) mitochondria, the concentrations of these enzymes were 9.5, 1.8, 2.0, 0.92, 1.5, and 2.1 times higher, respectively, than in the synaptosomes. The increase with age in succinate dehydrogenase and glutaminase was restricted to free mitochondria while hexokinase and malate NADP dehydrogenase accumulated and beta-hydroxybutyrate dehydrogenase diminished in both fractions. In adult brain, too, where the above ratios became 7.5, 5.2, 3.5, 0.84, 1.4, and 2.0, respectively, the concentrations of enzymes relative to each other distinguished clearly between free and synaptic mitochondria. The results substantiate previously noted signs of mitochondrial heteroeneity in adult brain, and extend them to immature brain. The chemical composition, the quantitative pattern of enzymes, of free and synaptic mitochondria is clearly different, and undergoes separate changes during postnatal differentiation.

The role of coenzymes, cortisone and RNA in the control of liver enzyme levels

The tryptophan-induced elevation of rat liver tryptophan pyrrolase activity in vivo is associated with two consecutive processes: (a) saturation of the endogenous apotryptophan pyrrolase with its heme cofactor and (b) an increase in the amount of the protein moiety of the enzyme system which can be determined by immunological titration. Process (b) is inhibited by the administration of puromycin (an inhibitor of protein synthesis) but not by that of actinomycin (an inhibitor of RNA synthesis). The induction of tryptophan pyrrolase by cortisone involves process (b alone and is inhibited by puromycin as well as by actinomycin. These agents also inhibit the cortisone-induced elevation of the level of liver tyrosine-α-ketoglutarate transaminase. The results suggest a basic difference between the mechanism of substrate- and hormone-induced stimulation of enzyme synthesis. n nThe level of activity of rat liver threonine dehydrase is decreased by a vitamin B6 deficient diet. The degree of saturation of this enzyme with pyridoxal phosphate and the activity measured in the presence of excess cofactor is not altered by the administration of threonine in vivo. n nThe early postnatal accumulation of tryptophan pyrrolase, unlike that of tyrosine-α-ketoglutarate transaminase, is not inhibited by adrenalectomy or actinomycin. Observations on the ratio of holo- to apotryptophan pyrrolase during development also suggests a common mechanism underlying the developmental and substrate-induced elevation of this enzyme. n nThe present findings exemplify two ways in which factors regulating enzyme synthesis in adult or developing tissues may operate. They may influence the number of potential synthetic sites (RNA template units) or the speed of functioning of a constant number of these sites.

Distribution of glutamine hexosephosphate aminotransferase in rat tissues; changes with state of differentiation

Abstract The activity of glutamine hexosephosphate aminotransferase ( L -glutamine: D -fructose 6-phosphate aminotransferase, EC 2.6.1.16) was determined with an improved assay method in some three dozen rat tissues: adult, developing and neoplastic. The highest activities (20–200 units/g) were seen in colon, mammary (during late lactation), submaxillary, sublingual and parotid glands, placenta and liver. The activity increased strikingly along the length of the intestine; glucose feeding inhibited it in ileum and colon. In liver and intestine the activity increased with age but in brain, muscle, heart and kidney the activity was considerably higher during fetal (7.1–12.8 units/g) than in adult life (0.8–3.5 units/g). Renal, mammary and muscle tumors (but not hepatomas) had much higher activities (4–20.5 units/g) than the cognate normal adult tissue. The distribution pattern among tissues indicates that glutamine hexosephosphate aminotransferase is of general importance to all growing, undifferentiated tissues and of special importance to the differentiated function of particular adult organs. The latter are organs which engage in glycoprotein secretion. The results support the assumption that glutamine hexosephosphate aminotransferase activity is essential for glycoprotein synthesis.

Human colon tumors. Enzymic and histological characteristics

In samples of colonic adenocarcinomas, the mean activities of thymidine kinase, glucose‐6‐phosphate dehydrogenase, phosphoserine phosphatase and pyrroline‐5‐carboxylate reductase were several fold higher than those of non‐neoplastic colon. The presence of considerable, told labile pyrroline‐5‐carboxylate reductase activity provided an additional criterion for distinguishing tumors from the control tissue. Deviations from the pattern of enzymes in normal colon were much more pronounced in the five moderately well‐differentiated than in the single well‐differentiated adenocarcinoma.

Effects of hormones on development of fetal enzymes.

The adaptation of newborn mammals to extrauterine life is conditioned, to a large extent, by the maturity that their biochemical functions have attained during prenatal tissue differentiation. We concentrated on studying the development of enzyme systems in rat liver during late fetal and neonatal life; this was combined with the morphometry of cell types and of subcellular organelles. A cluster of enzymes that emerge during late fetal life and attain their adult concentrations at term has been distinguished from another cluster that makes its appearance immediately after birth. Different hormones (e.g., thyroxine, glucagon, and glucocorticoids), the secretion of which is the natural stimulus for the expression of specific groups of genes, have been identified. With the administration of these hormones to fetuses it is possible to evoke the synthesis of chosen enzymes before the scheduled time and to produce newborn mammals (delivered at term or prematurely) with precociously mature enzyme profiles. The potential medical application of the numerous experimental means by which we can now manipulate the course of enzymic differentiation remains to be evaluated.

Hydrocortisone regulation of thymidine kinase in thymus involution and hematopoietic tissues.

Abstract Thymus involution, initiated by hydrocortisone treatment, was accompanied by a 90% decrease of thymidine kinase activity per g tissue. The concentration of six other enzymes remained essentially unchanged and that of two increased significantly, indicating that hydrocortisone has a selective effect on the enzymic composition of this organ. Hydrocortisone also decreased the level of thymidine kinase in two erythropoietic organs, fetal liver and neonatal spleen. These results indicate a close connection between thymidine kinase and the effect of glucocorticoids on organs involved in blood cell turnover. The effect of hydrocortisone on thymidine kinase levels does not necessarily coincide with that on cellularity. In spleen of 3–4-day-old rats the hormone did not alter the number of nuclei per g. In fetal liver an injection of hydrocortisone can cause maximal loss of thymidine kinase before there is any detectable loss in the number of hematopoietic cells. These results suggest that intracellular changes in enzyme concentrations precede alterations in cytocomposition.

The regulation of apoenzyme levels by coenzymes and hormones

Summary Evidence has been obtained that coenzyme levels, in addition to regulating the activity of existing enzyme, may influence in vivo the amount of protein moiety of appropriate enzyme systems. Pyridoxine administration to rats (intact or adrenalectomized) causes, within 4 hr, an approximately 200 per cent increase in the level of liver tyrosine transaminase. This change reflects an increase in the concentration of the apoenzyme since the activity both before and after pyridoxine treatment was measured in the presence of excess added pyridoxal phosphate. Furthermore, puromycin, an inhibitor of protein synthesis, interferes with this cofactor-induced rise in vivo . The effectiveness of tryptophan analogues as inducers of rat liver tryptophan pyrrolase correlates with their ability to raise the holoto apotryptophan pyrrolase ratio. This provides further support for the hypothesis regarding the role of the heme coenzyme in the regulation of the synthesis of this enzyme. The possible mechanisms involved in cofactor induction are discussed and contrasted with the hormonal regulation of enzyme levels.